Alterations in cortical excitation and inhibition in genetic mouse models of Huntington's disease.

1Mental Retardation Research Center, David Geffen School of Medicine, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, Los Angeles, CA 90095, USA.

Abstract

Previously, we identified progressive alterations in spontaneous EPSCs and IPSCs in the striatum of the R6/2 mouse model of Huntington's disease (HD). Medium-sized spiny neurons from these mice displayed a lower frequency of EPSCs, and a population of cells exhibited an increased frequency of IPSCs beginning at approximately 40 d, a time point when the overt behavioral phenotype begins. The cortex provides the major excitatory drive to the striatum and is affected during disease progression. We examined spontaneous EPSCs and IPSCs of somatosensory cortical pyramidal neurons in layers II/III in slices from three different mouse models of HD: the R6/2, the YAC128, and the CAG140 knock-in. Results revealed that spontaneous EPSCs occurred at a higher frequency, and evoked EPSCs were larger in behaviorally phenotypic mice whereas spontaneous IPSCs were initially increased in frequency in all models and subsequently decreased in R6/2 mice after they displayed the typical R6/2 overt behavioral phenotype. Changes in miniature IPSCs and evoked IPSC paired-pulse ratios suggested altered probability of GABA release. Also, in R6/2 mice, blockade of GABA(A) receptors induced complex discharges in slices and seizures in vivo at all ages. In conclusion, altered excitatory and inhibitory inputs to pyramidal neurons in the cortex in HD appear to be a prevailing deficit throughout the development of the disease. Furthermore, the differences between synaptic phenotypes in cortex and striatum are important for the development of future therapeutic approaches, which may need to be targeted early in the development of the phenotype.

A: Typical voltage clamp traces are shown for both WT and R6/2 cells from mice in each age group. B: CNQX/AP5 application completely blocked all synaptic currents at a membrane holding potential of −70 mV. Insets show traces on a magnified time scale to more clearly show the effect of blocking ionotropic glutamate receptors. C: Mean frequencies (±S.E.M.) of EPSCs recorded in ACSF for each age group. Note the significant decrease in frequency with age in cells from WTs that does not occur in cells from R6/2 mice. D: Amplitude-frequency histograms for EPSCs recorded in ACSF for each age group. E: Cumulative probability curves for inter-event interval showing a significant increase in release probability at 80 days. In this and subsequent figures, sample sizes are in parentheses and significant differences are denoted by * p<0.05; ** p<0.01; *** p<0.001; † p<0.05-p<0.001.

A: Traces show examples of average EPSCs with an exponential fit for WT (black) and R6/2 (grey) cells at each age (n>100 per trace). B: Bar graphs show rise times, decay times and half-amplitude durations of spontaneous EPSCs at each age. Note the progressive decrease in decay times and half-amplitude durations of spontaneous EPSCs of R6/2 groups at 40 and 80 days.

A: Example traces showing EPSCs recorded in the presence of BIC (20 μM) for each age group. B: Mean frequency of EPSCs (±S.E.M.) for each group. C: Mean percent change in frequency from ACSF to BIC for each group. D: Amplitude-frequency histograms revealed a greater frequency of small amplitude (5–10 pA) currents at 80 days. E: Cumulative inter-event interval plots for each group, showing an increased probability of release in cells from R6/2 mice at 80 days.

Miniature EPSCs in slices from 80 day mice were isolated with TTX. A: Typical traces from cells from WT and R6/2 mice, prior to and following the application of TTX. B: Amplitude-frequency histograms revealed that the increases in spontaneous EPSC frequency were maintained in TTX and were therefore independent of action potentials. C: No change in cumulative amplitude plots but a greater probability of release was apparent in cumulative frequency probability plots in the presence of TTX.

A: Voltage clamp recordings show that application of BIC induced large amplitude discharges in both genotypes. R6/2 cells showed more complex wave forms than cells from WT mice at each age. B: Proportion of cells displaying large amplitude discharges (left). Mean frequencies of large amplitude discharges for both genotypes at the three ages (middle). The percentage of cells showing complex discharges was greater in R6/2 mice and became more frequent with age (right). C: Distribution histograms for the duration of complex discharges. D. Current clamp recordings of cells from 80 day mice also showed more complex discharges in cells from R6/2 mice compared to those from WTs.

A: Typical recordings of spontaneous (s) and miniature (m) IPSCs in cells voltage clamped at +10 mV. CNQX and AP5 were present throughout the recordings, except at 80 days in the presence of TTX. At 21 days, two populations of cells were evident: a low frequency (LF) group and high frequency (HF) group. Note the bursting pattern of activity in the HF group of cells from R6/2 mice. At 40 and 80 days, only one population could be identified. B: Cumulative frequency probability plots of sIPSCs. Insets: Mean frequency of IPSCs (±S.E.M.) for each age group. C: Cumulative amplitude probability plots. Note: Abscissa in panel B is plotted on different time scales in order to better illustrate the differences between genotypes.

A: Traces show examples of average IPSCs with an exponential fit for WT and R6/2 cells at each age (n>100 per trace). Inset shows the different kinetics of IPSCs recorded in the HF group of R6/2 cells at 21 days. As the decay kinetics do not fit an exponential function, analyses for this group were not performed. B: Bar graphs show rise times, decay times and half-amplitude durations of spontaneous EPSCs at each age. Note the increase in decay times and half-amplitude durations of spontaneous IPSCs of R6/2 groups at 80 days.

A: Cumulative frequency probability plots showing that TTX abolishes differences in frequency at 21 days, but not 80 days. B: No differences were identified in cumulative amplitude probability plots at 21 days, but differences were maintained at 80 days. Traces in the presence of TTX are shown in .

A: Typical GABA-evoked responses in dissociated pyramidal neurons from WT (black) and R6/2 (grey) mice at each age and at 10 and 1000 μM GABA. Note that at 80 days, amplitudes are reduced in neurons from R6/2s. B: Peak current densities at each age. Note that normalizing cells to capacitance eliminates the difference in current amplitude. C: GABA desensitization times in neurons from 40 day animals. D: Zolpidem modulation at 40 days. Typical traces from WT and R6/2 cells before and after zolpidem application.

R6/2 mice were more susceptible to seizures induced by systemic administration of the GABAA receptor antagonist picrotoxin. Significant deceases in latencies occurred at all ages for both clonic and tonic-clonic seizures.

Cortical spontaneous synaptic currents in 6 (left) and 12 (middle) month YAC128 (white bars) and 12 month CAG140 KI mice (right; grey bars) and their respective WTs littermates (black bars in all graphs). A: Spontaneous EPSCs were recorded at a membrane potential of −70mV. Distributions show amplitude-frequency histograms for EPSCs recorded in ACSF for each group. Insets show average frequencies. B: Cell membranes were voltage clamped at +10mV and spontaneous IPSCs isolated using CNQX and AP5. Distributions show amplitude-frequency histograms for IPSCs for each group. Insets show average frequencies.